L.M.E. is supported by the DFG research training program GRK2157 entitled &#8220;3D Tissue Models for Studying Microbial Infections by Human Pathogens&#8221; awarded to A. S-U. B.J.K. is supported by a postdoctoral fellowship by the Alexander von Humboldt Foundation. Additionally, we acknowledge Lena Wolter for her technical assistance in the generation of the iPSC-BECs in culture.

<ol><li>Rua, R., McGavern, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Advances+in+Meningeal+Immunity%5BTitle%5D)+AND+%22Trends+in+Molecular+Medicine%22%5BJournal%5D)">Advances in Meningeal Immunity.</a> Trends in Molecular Medicine. 24 (6), 542-559 (2018).</li>
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<li>Slanina, H., Hebling, S., Hauck, C. R., Schubert-Unkmeir, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+invasion+by+neisseria+meningitidis+requires+a+functional+interplay+between+the+focal+adhesion+kinase%2C+Src+and+cortactin%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">Cell invasion by neisseria meningitidis requires a functional interplay between the focal adhesion kinase, Src and cortactin.</a> PLoS ONE. 7 (6), (2012).</li>
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<li>Vatine, G. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modeling+Psychomotor+Retardation+using+iPSCs+from+MCT8-Deficient+Patients+Indicates+a+Prominent+Role+for+the+Blood-Brain+Barrier%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">Modeling Psychomotor Retardation using iPSCs from MCT8-Deficient Patients Indicates a Prominent Role for the Blood-Brain Barrier.</a> Cell Stem Cell. , (2016).</li>
<li>Lim, R. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Huntington%27s+Disease+iPSC-Derived+Brain+Microvascular+Endothelial+Cells+Reveal+WNT-Mediated+Angiogenic+and+Blood-Brain+Barrier+Deficits%5BTitle%5D)+AND+%22Cell+Reports%22%5BJournal%5D)">Huntington's Disease iPSC-Derived Brain Microvascular Endothelial Cells Reveal WNT-Mediated Angiogenic and Blood-Brain Barrier Deficits.</a> Cell Reports. 19 (7), 1365-1377 (2017).</li>
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<li>Wilson, H. K., Faubion, M. G., Hjortness, M. K., Palecek, S. P., Shusta, E. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cryopreservation+of+brain+endothelial+cells+derived+from+human+induced+pluripotent+stem+cells+is+enhanced+by+rho-associated+coiled+coil-containing+kinase+inhibition%5BTitle%5D)+AND+%22Tissue+Engineering+-+Part+C%3A+Methods%22%5BJournal%5D)">Cryopreservation of brain endothelial cells derived from human induced pluripotent stem cells is enhanced by rho-associated coiled coil-containing kinase inhibition.</a> Tissue Engineering - Part C: Methods. , (2016).</li>
<li>Qian, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Directed+differentiation+of+human+pluripotent+stem+cells+to+blood-brain+barrier+endothelial+cells%5BTitle%5D)+AND+%22Science+Advances%22%5BJournal%5D)">Directed differentiation of human pluripotent stem cells to blood-brain barrier endothelial cells.</a> Science Advances. , (2017).</li>
<li>Sances, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+iPSC-Derived+Endothelial+Cells+and+Microengineered+Organ-Chip+Enhance+Neuronal+Development%5BTitle%5D)+AND+%22Stem+Cell+Reports%22%5BJournal%5D)">Human iPSC-Derived Endothelial Cells and Microengineered Organ-Chip Enhance Neuronal Development.</a> Stem Cell Reports. , (2018).</li>
<li>Canfield, S. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+isogenic+blood-brain+barrier+model+comprising+brain+endothelial+cells%2C+astrocytes%2C+and+neurons+derived+from+human+induced+pluripotent+stem+cells%5BTitle%5D)+AND+%22Journal+of+Neurochemistry%22%5BJournal%5D)">An isogenic blood-brain barrier model comprising brain endothelial cells, astrocytes, and neurons derived from human induced pluripotent stem cells.</a> Journal of Neurochemistry. , (2017).</li>
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</ol>

The authors have nothing to disclose.

Modeling BECs and the BBB has had challenges, as primary and immortalized human BECs, in vitro, tend to lack robust barrier phenotypes. The advent of human&#160;stem cell technologies has&#160;allowed for the generation of&#160;iPSC derived BEC-like cells&#160;that retain expected hallmark&#160;BBB phenotypes such as&#160;endothelial markers, tight&#160;junction expression, barrier&#160;properties, response to other&#160;CNS cell types, and functional&#160;efflux transporters12,</sup...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/61400">Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells Larvae</a>. The Authors section was updated from:
Leo M. Endres1 
Alexandra Schubert-Unkmeir1 
Brandon J. Kim1,2 
1Institute for Hygiene and Microbiology, University of W&#252;rzburg 
2Department of Biological Sciences, University of Alabama
to:
Leo M. Endres1 
Sarah F. Hathcock2 
Alexandra Schubert-Unkmeir1 
Brandon J. Kim1,2 
1Institute for Hygiene and Microbiology, University of W&#252;rzburg 
2Department of Biological Sciences, University of Alabama

An erratum was issued for:&#160;<a href="https://www.jove.com/t/61400">Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells Larvae</a>. The Authors section was updated.

Erratum: Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells

The blood-brain barrier (BBB), and the meningeal blood-CSF barrier (mBCSFB) are extremely tight cellular barriers that separate the circulation from the central nervous system (CNS) and are primarily comprised of highly specialized brain endothelial cells (BECs)1,2. Together, BECs maintain proper brain homeostasis by regulating nutrients and waste products in and out of the brain, while excluding many toxins, drugs, and pathogens1,2. Bacterial meningitis occurs when blood-borne bacteria are able to interact with, and penetrate the barrier formed by BECs and cause inflammation. Neisseria meningitidis (Nm, meningococcus) is a Gram-negative bacterium that colonizes the nasopharaynx of 10&#8210;40 % of healthy individuals, but in some cases can cause serious systemic disease3. In affected individuals, Nm can gain access to the blood stream where it can cause purpura fulminans as well as penetrate BECs gaining access to the CNS causing meningitis3. Nm is a leading cause of bacterial meningitis world-wide, and despite vaccination efforts, is still a primary cause of meningitis4. Modern medical intervention, such as antibiotic treatment, have made these conditions survivable, however those affected with meningitis often are left with permanent neurological damage5,6.
Previous studies have identified bacterial factors and host signaling that contribute to Nm-BEC interactions7,8,9,10,11. The identified adhesins and invasins such as the opacity protein Opc, and type-IV pili, as well as receptors such as CD147, have been conducted on various BEC models in vitro, however these models lack many defining BBB properties7,9,11,12. Complete understanding of Nm-BEC interactions remain elusive due partially to the inability to utilize in vivo models, incomplete vaccination protection, and lack of robust human BEC models in vitro.
Modeling hBECs in vitro has been challenging due to the unique properties of BECs. Compared with peripheral endothelial cells, BECs have a number of phenotypes that enhance their barrier properties such as high trans-endothelial electrical resistance (TEER) due to complex tight junctions12. Once removed from the brain microenvironment, BECs rapidly lose their barrier properties limiting the usefulness of primary or immortalized in vitro models that only form a weak barrier12,13. The combination of the human specificity of Nm infections, lack of robust in vivo models, and challenges modeling human BECs in vitro creates a need for better models to understand the complex host-pathogen interaction between Nm and BECs. Recently using model human&#160;induced pluripotent stem cell&#160;(iPSC) technologies BEC-like&#160;cells have been derived from&#160;iPSCs that better mimic BECs&#160;in vivo12,13,14,15. iPSC-BECs are of human origin, easily scalable, and possess expected BEC phenotypes compared to their primary or immortalized counterparts12,13,14,15. Additionally we and others have demonstrated that iPSC-BECs are useful for modeling various diseases of the CNS such as host-pathogen interaction, Huntington&#8217;s disease, and MCT8 deficiency that causes Allan-Hurndon-Dudley syndrome16,17,18,19,20,21. Here, we demonstrate how to derive iPSC-BECs from renewable iPSC sources and the infection of iPSC-BECs with Nm leading to activation of the innate immune response. We believe that this model is useful to interrogate host-pathogen interaction that is unable to be recapitulated in other in vitro models and is especially useful when examining interactions with human specific pathogens such as Nm.

<table><tbody><tr><td>Accutase (1x)</td><td>Sigma</td><td>A6964</td><td>Enzymatic cell dissociation reagent</td></tr><tr><td>Acetic acid</td><td>Sigma</td><td>A6283</td><td></td></tr><tr><td>All-trans retinoic acid (RA)</td><td>Sigma</td><td>R2625</td><td></td></tr><tr><td>Anti-CD31 (PECAM-1)</td><td>Thermo Scientific (Labvision)</td><td>RB-10333</td><td></td></tr><tr><td>Anti-Claudin-5</td><td>Invitrogen</td><td>4C3C2</td><td></td></tr><tr><td>Anti-Glut-1</td><td>Thermo Scientific (Labvision)</td><td>SPM498 (MA5-11315)</td><td></td></tr><tr><td>Anti-Occludin</td><td>Invitrogen</td><td>33-1500</td><td></td></tr><tr><td>Anti-VE-cadherin</td><td>Santa Cruz</td><td>sc-52751</td><td></td></tr><tr><td>Anti-ZO-1</td><td>Invitrogen</td><td>33-9100</td><td></td></tr><tr><td>Bacto Proteose Peptone</td><td>BD</td><td>211684</td><td></td></tr><tr><td>b-Mercaptoethanol</td><td>Merck (Sigma-Aldrich)</td><td>805740</td><td></td></tr><tr><td>Cell culture plates and flasks</td><td>Sarstedt</td><td></td><td></td></tr><tr><td>Centrifuge (Heraeus Megafuge 1.0R)</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Class II biosafety cabinet</td><td>Nuaire</td><td>NU-437-400E</td><td></td></tr><tr><td>CO2 Incubator (DHD Autoflow CO2 Air-Jacketed Incubator)</td><td>Nuaire</td><td></td><td></td></tr><tr><td>Collagen IV</td><td>Sigma</td><td>C5533</td><td></td></tr><tr><td>Columbia ager + 5 % sheep blood</td><td>Biomerieux</td><td>43049</td><td></td></tr><tr><td>Costar Transwell polyester filters (12- or 24-well)</td><td>Corning</td><td>3460, 3470</td><td></td></tr><tr><td>D(+)-Glucose</td><td>Merck (Sigma-Aldrich)</td><td>G8270</td><td></td></tr><tr><td>DAPI</td><td>Invitrogen</td><td>D1306</td><td></td></tr><tr><td>DMEM/F12</td><td>Gibco</td><td>31330-038</td><td></td></tr><tr><td>DMSO</td><td>ROTH</td><td>A994.1</td><td></td></tr><tr><td>Dulbecco&#039;s phosphate-buffered saline (DPBS)</td><td>Gibco</td><td>21600-069</td><td></td></tr><tr><td>Epithelial Volt-Ohm Meter (Millicell ERS-2) with STX electrode</td><td>Merck (Millipore)</td><td>MERS00002</td><td></td></tr><tr><td>Fe(NO&lt;sub&gt;3&lt;/sub&gt;)&lt;sub&gt;3&lt;/sub&gt;</td><td>ROTH</td><td>5632.1</td><td></td></tr><tr><td>Fibronectin</td><td>Sigma</td><td>F1141</td><td></td></tr><tr><td>Fluoresence microscope (Eclipse Ti)</td><td>Nikon</td><td></td><td></td></tr><tr><td>Hemacytometer (Neubauer)</td><td>A. Hartenstein</td><td>ZK06</td><td></td></tr><tr><td>Human basic fibroblast growth factor (bFGF)</td><td>PeproTech</td><td>100-18B</td><td></td></tr><tr><td>Human Endothelial Serum Free Medium (hESFM)</td><td>Gibco</td><td>11111-044</td><td></td></tr><tr><td>Inverted microscope (Wilovert)</td><td>Hund (Will Wetzlar)</td><td></td><td></td></tr><tr><td>iPS(IMR90)-4 cells</td><td>WiCell</td><td></td><td></td></tr><tr><td>Kellogg&#039;s supplement</td><td></td><td></td><td>To prepare 110 ml of Kellogg&#039;s supplement, prepare 100 ml of 4 g/ml glucose, 0.1 g/ml glutamine, and 0.2 mg/ml thiamine pyrophosphate and 10 ml of 5 mg/ml Fe(NO&lt;sub&gt;3&lt;/sub&gt;)&lt;sub&gt;3&lt;/sub&gt; and combine the solutions. Filter sterilize and store aliquoted at -20 &amp;deg;C.</td></tr><tr><td>Knockout serum replacement (KOSR)</td><td>Gibco</td><td>10828-028</td><td></td></tr><tr><td>L-glutamine (GlutaMAX)</td><td>Invitrogen</td><td>35050-038</td><td></td></tr><tr><td>LunaScript RT SuperMix Kit</td><td>NEB</td><td>E3010L</td><td>cDNA synthesis kit</td></tr><tr><td>Matrigel Matrix</td><td>Corning</td><td>354230</td><td></td></tr><tr><td>Methanol</td><td>ROTH</td><td>4627.5</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>ROTH</td><td>KK36.1</td><td></td></tr><tr><td>Micropipettes (Research Plus)</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>ROTH</td><td>6329</td><td></td></tr><tr><td>Nonessential amino acids (NEAA)</td><td>Gibco</td><td>11140-035</td><td></td></tr><tr><td>NucleoSpin RNA isolation kit</td><td>Machery-Nagel</td><td>740955</td><td>RNA isolation kit</td></tr><tr><td>Pipette boy (Accu-Jet Pro)</td><td>Brand</td><td></td><td></td></tr><tr><td>Platelet poor plasma-derived serum, bovine (PDS)</td><td>Fisher</td><td>50-443-029</td><td></td></tr><tr><td>PowerUp SYBR Green Master Mix</td><td>Applied Biosystems</td><td>A25742</td><td>qPCR master mix</td></tr><tr><td>qPCR film (MicroAmp Optical Adhesive Film)</td><td>Applied Biosystems</td><td>4211971</td><td></td></tr><tr><td>qPCR plates (MicroAmp Fast 96-well)</td><td>Applied Biosystems</td><td>4346907</td><td></td></tr><tr><td>ROCK inhibitor, Y27632 dihydrochloride</td><td>Tocris</td><td>1254</td><td></td></tr><tr><td>RT-PCR thermo cycler (StepOnePlus)</td><td>Applied Biosystems</td><td>4376600</td><td></td></tr><tr><td>Serological pipettes</td><td>Sarstedt</td><td></td><td></td></tr><tr><td>StemFlex basal medium + 50x StemFlex supplement</td><td>Gibco</td><td>A3349401</td><td>Stem-cell maintenance medium</td></tr><tr><td>Swinging Bucket Rotor (Heraeus #2704)</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Thiamine pyrophosphate</td><td>Sigma</td><td>C8754-5G</td><td></td></tr><tr><td>Trypan Blue Solution, 0.4%</td><td>Gibco</td><td>15250061</td><td></td></tr><tr><td>Versene</td><td>Gibco</td><td>15040-033</td><td>Non-enzymatic cell dissociation reagent (EDTA)</td></tr></tbody></table>

neisseria meningitidis infection of induced pluripotent stem-cell derived brain endothelial cells

Meningococcal meningitis is a life-threatening infection that occurs when Neisseria meningitidis (meningococcus, Nm) can gain access to the central nervous system (CNS) by penetrating highly specialized brain endothelial cells (BECs). As Nm is a human-specific pathogen, the lack of robust in vivo model systems makes study of the host-pathogen interactions between Nm and BECs challenging and establishes a need for a human based model that mimics native BECs. BECs possess tighter barrier properties when compared to peripheral endothelial cells characterized by complex tight junctions and elevated trans-endothelial electrical resistance (TEER). However, many in vitro models, such as primary BECs and immortalized BECs, either lack or rapidly lose their barrier properties after removal from the native neural microenvironment. Recent advances in human stem-cell technologies have developed methods for deriving brain-like endothelial cells from induced pluripotent stem-cells (iPSCs) that better phenocopy BECs when compared to other in vitro human models. The use of iPSC-derived BECs (iPSC-BECs) to model Nm-BEC interaction has the benefit of using human cells that possess BEC barrier properties, and can be used to examine barrier destruction, innate immune activation, and bacterial interaction. Here we demonstrate how to derive iPSC-BECs from iPSCs in addition to bacterial preparation, infection, and sample collection for analysis.

The protocol described here is adapted from Stebbins et al. and highlights the process to differentiate iPSCs into brain-like endothelial cells that possess BBB properties, and how to utilize this model for infection studies using iPSC-BECs with Nm19,22. The iPSC-BECs, when differentiated properly, exhibit tight barrier properties measured by TEER that are often greater than 2000 &#937;&#183;cm2, and express endothelial markers such as VE-cadherin and ...

Watch this Scientific Journal Video about Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells at JoVE.com

Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells

The protocol described here highlights the major steps in the differentiating induced pluripotent stem-cell derived brain-like endothelial cells, the preparation of Neisseria meningitidis for infection, and sample collection for other molecular analyses.

Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells

Meningococcal meningitis is a life-threatening infection that occurs when Neisseria meningitidis (meningococcus, Nm) can gain access to the central ...

neisseria-meningitidis-infection-induced-pluripotent-stem-cell

Research

JoVE Journal

Immunology and Infection

2.4K Views.  University of Würzburg. The protocol described here highlights the major steps in the differentiating induced pluripotent stem-cell derived brain-like endothelial cells, the preparation of Neisseria meningitidis for infection, and sample collection for other molecular analyses.

Video: Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells

Generation of a Human iPSC-Based Blood-Brain Barrier Chip

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the blood that can disrupt delicate brain function. As such, the BBB is a major obstacle to the delivery of therapeutics to the CNS. Accumulating evidence suggests that the BBB plays a key role in the onset and progression of neurological diseases. Thus, there is a tremendous need for a BBB model that can predict penetration of CNS-targeted drugs as well as elucidate the BBB&#39;s role in health and disease.
We have recently combined organ-on-chip and induced pluripotent stem cell (iPSC) technologies to generate a BBB chip fully personalized to humans. This novel platform displays cellular, molecular, and physiological properties that are suitable for the prediction of drug and molecule transport across the human BBB. Furthermore, using patient-specific BBB chips, we have generated models of neurological disease and demonstrated the potential for personalized predictive medicine applications. Provided here is a detailed protocol demonstrating how to generate iPSC-derived BBB chips, beginning with differentiation of iPSC-derived brain microvascular endothelial cells (iBMECs) and resulting in mixed neural cultures containing neural progenitors, differentiated neurons, and astrocytes. Also described is a procedure for seeding cells into the organ chip and culturing of the BBB chips under controlled laminar flow. Lastly, detailed descriptions of BBB chip analyses are provided, including paracellular permeability assays for assessing drug and molecule permeability as well as immunocytochemical methods for determining the composition of cell types within the chip.

The blood-brain barrier (BBB) is a multicellular neurovascular unit tightly regulating brain homeostasis. By combining human iPSCs and organ-on-chip technologies, we have generated a personalized BBB chip, suitable for disease modeling and CNS drug penetrability predictions. A detailed protocol is described for the generation and operation of the BBB chip.

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the ...

Developmental Biology

Generating Human Brain Organoids with Resident Microglia Using iPSC-Derived HPCs

Derivation of a Human Brain Organoid with Microglia Development

Three-dimensional (3D) brain organoid cultures derived from induced pluripotent stem cells (iPSC) provide an important alternative in vitro tool&#160;for studying human brain development and pathogenesis of neurological diseases. However, the lack of incorporation of microglia in the human brain organoids is still a major hurdle for 3D models of neuroinflammation. Current approaches include either the incorporation of fully differentiated microglia into mature brain organoids or the induction of microglial differentiation from the early stage of iPSC-derived embryoid bodies (EBs). The first approach misses the stage when microglial differentiation interacts with the adjacent neural environment, and the later approach is technically challenging, resulting in inconsistency among the final organoids in terms of the quantity and quality of microglia. To model brain organoids with microglia to study the early interactions between microglial and neuronal development, highly pure hematopoietic progenitor cells (HPC) differentiated from human iPSCs were incorporated&#160;into iPSC-derived EBs to make brain organoids. Using immunostaining and single-cell RNA sequencing (sc-RNA-seq) analysis, we confirmed that HPCs were incorporated into the 3D organoids, which eventually developed into brain organoids with both microglia and neurons. Compared to brain organoids without HPCs, this approach produces significant microglial incorporation in the brain organoids. This novel 3D organoid model, which consists of both microglial and neural development properties, can be used to study the early interactions between innate immune and nervous system development and potentially as a model for neuroinflammation and neuroinfectious disorders.

We present a protocol to generate a human brain organoid with resident microglia by incorporating Induced pluripotent stem cell (iPSC)-derived hematopoietic progenitor cells (HPCs) into organoid development.

Three-dimensional (3D) brain organoid cultures derived from induced pluripotent stem cells (iPSC) provide an important alternative in vitro tool&#160;for ...

Inducing Meningococcal Meningitis Serogroup C in Mice via Intracisternal Delivery

Neisseria meningitidis (meningococcus) is a narrow-host-range microorganism, globally recognized as the leading cause of bacterial meningitis. Meningococcus is a transient colonizer of human nasopharynx of approximately 10% of healthy subject. In particular circumstances, it acquires an invasive ability to penetrate the mucosal barrier and invades the bloodstream causing septicaemia. In the latest case, fulminating sepsis could arise even without the consequent development of meningitis. Conversely, bacteria could poorly multiply in the bloodstream, cross the blood brain barrier, reach the central nervous system, leading to fulminant meningitis. The murine models of bacterial meningitis represent a useful tool to investigate the host-pathogen interactions and to analyze the pathogenetic mechanisms responsible for this lethal disease. Although, several experimental model systems have been evaluated over the last decades, none of these were able to reproduce the characteristic pathological events of meningococcal disease. In this experimental protocol, we describe a detailed procedure for the induction of meningococcal meningitis in a mouse model based on the intracisternal inoculation of bacteria. The peculiar signs of human meningitis were recorded in the murine host through the assessment of clinical parameters (e.g., temperature, body weight), evaluation of survival rate, microbiological analysis and histological examination of brain injury. When using intracisternal (i.cist.) inoculum, meningococci complete delivery directly into cisterna magna, leading to a very efficient meningococcal replication in the brain tissue. A 1,000-fold increase of viable count of bacteria is observed in about 18 h. Moreover, meningococci are also found in the spleen, and liver of infected mice, suggesting that the liver may represent a target organ for meningococcal replication.

Here, we describe a method to induce meningococcal meningitis through an intracisternal route of infection in adult mice. We present a step by step protocol of meningococcal infection from the preparation of inoculum to the intracisternal infection; then record the animal survival and evaluate the bacterial loads in murine tissues.

Neisseria meningitidis (meningococcus) is a narrow-host-range microorganism, globally recognized as the leading cause of bacterial meningitis. ...

Differentiation of Human Induced Pluripotent Stem Cells to Brain Microvascular Endothelial Cell-Like Cells with a Mature Immune Phenotype

Blood-brain barrier (BBB) dysfunction is a pathological hallmark of many neurodegenerative and neuroinflammatory diseases affecting the central nervous system (CNS). Due to the limited access to disease-related BBB samples, it is still not well understood whether BBB malfunction is causative for disease development or rather a consequence of the neuroinflammatory or neurodegenerative process. Human induced pluripotent stem cells (hiPSCs) therefore provide a novel opportunity to establish in vitro BBB models from healthy donors and patients, and thus to study disease-specific BBB characteristics from individual patients. Several differentiation protocols have been established for deriving brain microvascular endothelial cell (BMEC)-like cells from hiPSCs. Consideration of the specific research question is mandatory for the correct choice of the respective BMEC-differentiation protocol. Here, we describe the extended endothelial cell culture method (EECM), which is optimized to differentiate hiPSCs into BMEC-like cells with a mature immune phenotype, allowing the study of immune cell-BBB interactions. In this protocol, hiPSCs are first differentiated into endothelial progenitor cells (EPCs) by activating Wnt/&#946;-catenin signaling. The resulting culture, which contains smooth muscle-like cells (SMLCs), is then sequentially passaged to increase the purity of endothelial cells (ECs) and to induce BBB-specific properties. Co-culture of EECM-BMECs with these SMLCs or conditioned medium from SMLCs allows for the reproducible, constitutive, and cytokine-regulated expression of EC adhesion molecules. Importantly, EECM-BMEC-like cells establish barrier properties comparable to primary human BMECs, and due to their expression of all EC adhesion molecules, EECM-BMEC-like cells are different from other hiPSC-derived in vitro BBB models. EECM-BMEC-like cells are thus the model of choice for investigating the potential impact of disease processes at the level of the BBB, with an impact on immune cell interaction in a personalized fashion.

Here, we describe a protocol, the extended endothelial cell culture method (EECM), that allows differentiation of pluripotent stem cells to brain microvascular endothelial cell (BMEC)-like cells. These cells show endothelial cell adhesion molecule expression and are thus a human blood-brain barrier model suitable to study immune cell interactions in vitro.

Blood-brain barrier (BBB) dysfunction is a pathological hallmark of many neurodegenerative and neuroinflammatory diseases affecting the central nervous ...

Bioengineering

Induction of Leptomeningeal Cells Modification Via Intracisternal Injection

The protocol outlined here describes how to safely and manually inject solutions through the cisterna magna while eliminating the risk of damage to the underlying parenchyma. Previously published protocols recommend using straight needles that should be lowered to a maximum of 1-2 mm from the dural surface. The sudden drop in resistance once the dural membrane has been punctured makes it difficult to maintain the needle in a steady position. Our method, instead, employs a needle bent at the tip that can be stabilized against the occipital bone of the skull, thus preventing the syringe from penetrating into the tissue after perforation of the dural membrane. The procedure is straightforward, reproducible, and does not cause long-lasting discomfort in the operated animals. We describe the intracisternal injection strategy in the context of genetic fate mapping of vascular leptomeningeal cells. The same technique can, furthermore, be utilized to address a wide range of research questions, such as probing the role of leptomeninges in neurodevelopment and the spreading of bacterial meningitis, through genetic ablation of genes putatively implicated in these phenomena. Additionally, the procedure can be combined with an automatized infusion system for a constant delivery and used for tracking cerebrospinal fluid movement via injection of fluorescently labelled molecules.

We describe an intracisternal injection that employs a needle bent at the tip that can be stabilized to the skull, thus eliminating the risk of damage to the underlying parenchyma. The approach can be used for genetic fate mapping and manipulations of leptomeningeal cells and for tracking cerebrospinal fluid movement.

The protocol outlined here describes how to safely and manually inject solutions through the cisterna magna while eliminating the risk of damage to the ...

Neuroscience

Derivation, Expansion, Cryopreservation and Characterization of Brain Microvascular Endothelial Cells from Human Induced Pluripotent Stem Cells

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models for studying blood-brain barrier (BBB) function. This modified protocol provides detailed steps to derive, expand, and cryopreserve BMECs from human iPSCs using a different donor and reagents than those reported in previous protocols. iPSCs are treated with essential 6 medium for 4 days, followed by 2 days of human endothelial serum-free culture medium supplemented with basic fibroblast growth factor, retinoic acid, and B27 supplement. At day 6, cells are sub-cultured onto a collagen/fibronectin matrix for 2 days. Immunocytochemistry is performed at day 8 for BMEC marker analysis using CLDN5, OCLN, TJP1, PECAM1, and SLC2A1. Western blotting is performed to confirm BMEC marker expression, and absence of SOX17, an endodermal marker. Angiogenic potential is demonstrated with a sprouting assay. Trans-endothelial electrical resistance (TEER) is measured using chopstick electrodes and voltohmmeter starting at day 7. Efflux transporter activity for ATP binding cassette subfamily B member 1 and ATP binding cassette subfamily C member 1 is measured using a multi-plate reader at day 8. Successful derivation of BMECs is confirmed by the presence of relevant cell markers, low levels of SOX17, angiogenic potential, transporter activity, and TEER values ~2000 &#937; x cm2. BMECs are expanded until day 10 before passaging onto freshly coated collagen/fibronectin plates or cryopreserved. This protocol demonstrates that iPSC-derived BMECs can be expanded and passaged at least once. However, lower TEER values and poorer localization of BMEC markers was observed after cryopreservation. BMECs can be utilized in co-culture experiments with other cell types (neurons, glia, pericytes), in three-dimensional brain models (organ-chip and hydrogel), for vascularization of brain organoids, and for studying BBB dysfunction in neuropsychiatric disorders.

This protocol details an adapted method to derive, expand, and cryopreserve brain microvascular endothelial cells obtained by differentiating human induced pluripotent stem cells, and to study blood brain barrier properties in an ex vivo model.

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

Biology

Transplantation of Human Induced Pluripotent Stem Cell-Derived Microglia in Immunocompetent Mice Brain via Non-Invasive Transnasal Route

Microglia are the specialized population of macrophage-like cells of the brain. They play essential roles in both physiological and pathological brain functions. Most of our current understanding of microglia is based on experiments performed in the mouse. Human microglia differ from mouse microglia, and thus response and characteristics of mouse microglia may not always represent that of human microglia. Further, due to ethical and technical difficulties, research on human microglia is restricted to in vitro culture system, which does not capitulate in vivo characteristics of microglia. To overcome these issues, a simplified method to non-invasively transplant induced pluripotent stem cell-derived human microglia (iPSMG) into the immunocompetent mice brain via a transnasal route in combination with pharmacological depletion of endogenous microglia using a colony-stimulating factor 1 receptor (CSF1R) antagonist is developed. This protocol provides a way to non-invasively transplant cells into the mouse brain and may therefore be valuable for evaluating the in vivo role of human microglia in physiological and pathological brain functions.

The protocol presented here allows the transplantation of induced pluripotent stem cell-derived human microglia (iPSMG) into the brain via a transnasal route in immunocompetent mice. The method for the preparation and transnasal transplantation of cells and the administration of cytokine mixture for the maintenance of iPSMG is shown.

Microglia are the specialized population of macrophage-like cells of the brain. They play essential roles in both physiological and pathological brain ...

Intracisternal Delivery of Meningococci into Mouse Models to Induce Meningococcal Meningitis

Source: Pagliuca, C., et al., <a href="https://app.jove.com/v/60047">Inducing Meningococcal Meningitis Serogroup C in Mice via Intracisternal Delivery.</a> J. Vis. Exp. (2019)
In this video, we demonstrate the intracisternal delivery of Neisseria meningitidis into a mouse model to generate meningitis infection.

Source: Pagliuca, C., et al., Inducing Meningococcal Meningitis Serogroup C in Mice via Intracisternal Delivery. J. Vis. Exp. (2019)
In this video, we ...

JoVE Encyclopedia of Experiments

Immunology

Immune Response

Host-Pathogen Interaction

Generating Brain Endothelial Cells from Induced Pluripotent Stem Cells

Source: Endres, L. M., et al., <a href="https://app.jove.com/v/61400">Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells.</a> J. Vis. Exp. (2020)
This video demonstrates a protocol to induce the differentiation of induced pluripotent stem cells into brain endothelial cells.

1. Preparation of materials required for iPSC culture and BEC differentiation.

	Matrix coating of tissue culture (TC) plastic for IMR90-4 iPSC culture
	
	...

Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells

Summary

Explore More Videos

Generation of a Human iPSC-Based Blood-Brain Barrier Chip

Derivation of a Human Brain Organoid with Microglia Development

Inducing Meningococcal Meningitis Serogroup C in Mice via Intracisternal Delivery

Differentiation of Human Induced Pluripotent Stem Cells to Brain Microvascular Endothelial Cell-Like Cells with a Mature Immune Phenotype

Induction of Leptomeningeal Cells Modification Via Intracisternal Injection

Derivation, Expansion, Cryopreservation and Characterization of Brain Microvascular Endothelial Cells from Human Induced Pluripotent Stem Cells

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

Transplantation of Human Induced Pluripotent Stem Cell-Derived Microglia in Immunocompetent Mice Brain via Non-Invasive Transnasal Route

Intracisternal Delivery of Meningococci into Mouse Models to Induce Meningococcal Meningitis

Generating Brain Endothelial Cells from Induced Pluripotent Stem Cells